Microvascular density and hypoxia-inducible factor pathway in pancreatic endocrine tumours: negative correlation of microvascular density and VEGF expression with tumour progression

Abstract

Tumour-associated angiogenesis is partly regulated by the hypoxia-inducible factor (HIF) pathway. Endocrine tumours are highly vascularised and the molecular mechanisms of their angiogenesis are not fully delineated. The aim of this study is to evaluate angiogenesis and expression of HIF-related molecules in a series of patients with pancreatic endocrine tumours (PETs). The expression of vascular endothelial growth factor (VEGF), HIF-1alpha, HIF-2alpha and carbonic anhydrase 9 (CA9) was examined by immunohistochemistry in 45 patients with PETs and compared to microvascular density (MVD), endothelial proliferation, tumour stage and survival. Microvascular density was very high in PETs and associated with a low endothelial index of proliferation. Microvascular density was significantly higher in benign PETs than in PETs of uncertain prognosis, well-differentiated and poorly differentiated carcinomas (mean values: 535, 436, 252 and 45 vessels mm(-2), respectively, P < 0.0001). Well-differentiated tumours had high cytoplasmic VEGF and HIF-1alpha expression. Poorly differentiated carcinomas were associated with nuclear HIF-1alpha and membranous CA9 expression. Low MVD (P = 0.0001) and membranous CA9 expression (P = 0.0004) were associated with a poorer survival. Contrary to other types of cancer, PETs are highly vascularised, but poorly angiogenic tumours. As they progress, VEGF expression is lost and MVD significantly decreases. The regulation of HIF signalling appears to be specific in pancreatic endocrine tumours.

Figure 1

Immunohistochemical expression of VEGF, HIF-1α and HIF-2α by WHO-stage 1 (A, C, E and F) and WHO-stage 4 (B, D) PETs. (A, B) VEGF is highly expressed in a WHO stage-1 PET (A) and negative in a WHO-stage 4 PET (B). (C, D) HIF-1α cytoplasmic expression is strong in a WHO-stage 1 PET (C). HIF-1α nuclear expression is detected in a WHO-stage 4 PET (D). (E, F) HIF-2α cytoplasmic (E) and stromal (F) expression is detected in a WHO-stage 1 PET. Immunoperoxidase and haematoxylin nuclear counterstaining; original magnifications: A, × 250; B, × 250; C, × 150; D, × 500; E, × 200; F, × 250.

Figure 2

Box plot of VEGF cytoplasmic score stratified according to the WHO classification. Differences between groups were statistically significant (P=0.03).

Figure 3

Box plot of HIF-1α cytoplasmic score stratified according to the WHO classification. Differences between groups were statistically significant (P=0.008).

Figure 4

Box plot of HIF-1α nuclear expression stratified according to the WHO classification. Differences between groups were statistically significant (P=0.001).

Figure 5

Immunohistochemical expression of CA9 and CD34 by WHO-stage 1 (A, B) and WHO-stage 4 (C, D) pancreatic endocrine tumours and by VHL cases (E, F). (A, C, E) CA9 is not detected in most well-differentiated PETs (A). Its expression is strong and membranous around areas of necrosis in WHO-stage 4 PETs (C) and cytoplasmic diffuse in VHL cases (E). (B, D, F) CD34+ capillaries are numerous in WHO-stage 1 PETs (B) and scattered in WHO-stage 4 PETs (D). Microvascular density is very high in VHL cases (F). Immunoperoxidase and haematoxylin nuclear counterstaining; original magnifications: A, × 150; B, × 250; C, × 200; D, × 150; E, × 200; F, × 250.

Figure 6

Box plot of CA9 membranous expression stratified according to the WHO classification. Differences between groups were statistically significant (P=0.0001).

Figure 7

Survival curves according to Kaplan–Meier for 43 of the 45 patients of the study group according to CA9 membranous expression (thin line) or absence of expression (bold line); P=0.0004.

Figure 8

Box plot of MVD recorded in hotspots stratified according to the WHO classification. Differences between groups were statistically significant (P=0.0001).

Figure 9

Survival curves according to Kaplan–Meier for 43 of the 45 patients of the study group according to MVD <200 mm⁻² (thin line) or >200 mm⁻² (bold line); P=0.0001.